Bottom Line:
It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

Methodology/principal findings: In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.

Conclusions/significance: Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

pone-0036828-g003: N-acetylecysteine inhibits NRG-induced autophagy and cell death.(A) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM N-acetylcysteine (NAC) for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-p62 antibodies. Left panel, representative results. Right panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 6; means ± S.D; *p<0.05). (B) LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG in the presence or in the absence of 10 mM NAC. Methylene blue assay was performed 60 h later. Results are presented as % of control, and are the mean ± S.D of 4–6 determinations (**p<0.0001). This experiment was repeated three times with similar results. (C) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM NAC. Cells were harvested 60 h later and analyzed for their DNA content by flow cytometry. The percentage of cells at various cell cycle stages is indicated. (D) LNCaP cells were treated with 100 ng/ml NRG in the presence or in the absence of 10 mM NAC for 60 h. The cells were stained with the fluorescent DNA dye bisbenzimide (Hoecsht 33258, 1 µg/ml) to assess the number of dying cells. Following staining, the cells were photographed using Olympus optical inverted phase-contrast microscope Model IX70 (20×magnification; scale bars, 50 micrometer). Left panel, representative images. Right panel, percentage of dying cells was estimated by counting the number of Hoecsht-positive cells compared to the number total cells in each field (10–15 fields for each treatment, 100–200 cells per field). Results are presented as mean ± S.D (**p<0.0001).

Mentions:
It was previously demonstrated that autophagy induction depends on the formation and accumulation of ROS [23], [31], [32], [33]. Therefore, to characterize the effect of ROS on NRG-mediated autophagy and cell death, LNCaP cells were stimulated with NRG in the presence or in the absence of the general anti-oxidant N-acetylcysteine (NAC) [34], and LC3 and p62 levels were determined by immunoblot (Fig. 3A). Pre-incubation with NAC completely inhibited NRG-induced LC3-II elevation, indicating that NRG-induced autophagy is ROS-dependent. Next, we examined whether NAC can protect from NRG-induced cell death. LNCaP cells were pre-treated with NAC with and without NRG treatment, and cell viability was determined using the methylene blue staining assay. As demonstrated in Figure 3B, the presence of NAC prevented the decrease in cell viability induced by NRG. Two additional methods for detection of cell death (Hoecsht dye exclusion assay and flow cytometry) further supported these results. NRG induced enhanced cell death, as evident by the increase in sub-G1 population (Figure 3C) or by the high percentage of Hoecsht-positive cells (Figure 3D). This cell death was markedly inhibited by NAC. Furthermore, NAC treatment inhibited NRG-induced morphological change (S5). Hence, our findings clearly demonstrate that NAC inhibits the LC3-II accumulation, cell death and morphological changes induced by NRG in LNCaP cells.

pone-0036828-g003: N-acetylecysteine inhibits NRG-induced autophagy and cell death.(A) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM N-acetylcysteine (NAC) for 24 h. Whole cell lysates were prepared and subjected to an immunoblot analysis with anti-LC3 and anti-p62 antibodies. Left panel, representative results. Right panel, densitometric analysis is presented as fold induction over the control untreated cells (n = 6; means ± S.D; *p<0.05). (B) LNCaP cells were tested for cell viability using the methylene blue staining assay. Cells were treated with 100 ng/ml NRG in the presence or in the absence of 10 mM NAC. Methylene blue assay was performed 60 h later. Results are presented as % of control, and are the mean ± S.D of 4–6 determinations (**p<0.0001). This experiment was repeated three times with similar results. (C) LNCaP cells were treated with 100 ng/ml NRG with or without 10 mM NAC. Cells were harvested 60 h later and analyzed for their DNA content by flow cytometry. The percentage of cells at various cell cycle stages is indicated. (D) LNCaP cells were treated with 100 ng/ml NRG in the presence or in the absence of 10 mM NAC for 60 h. The cells were stained with the fluorescent DNA dye bisbenzimide (Hoecsht 33258, 1 µg/ml) to assess the number of dying cells. Following staining, the cells were photographed using Olympus optical inverted phase-contrast microscope Model IX70 (20×magnification; scale bars, 50 micrometer). Left panel, representative images. Right panel, percentage of dying cells was estimated by counting the number of Hoecsht-positive cells compared to the number total cells in each field (10–15 fields for each treatment, 100–200 cells per field). Results are presented as mean ± S.D (**p<0.0001).

Mentions:
It was previously demonstrated that autophagy induction depends on the formation and accumulation of ROS [23], [31], [32], [33]. Therefore, to characterize the effect of ROS on NRG-mediated autophagy and cell death, LNCaP cells were stimulated with NRG in the presence or in the absence of the general anti-oxidant N-acetylcysteine (NAC) [34], and LC3 and p62 levels were determined by immunoblot (Fig. 3A). Pre-incubation with NAC completely inhibited NRG-induced LC3-II elevation, indicating that NRG-induced autophagy is ROS-dependent. Next, we examined whether NAC can protect from NRG-induced cell death. LNCaP cells were pre-treated with NAC with and without NRG treatment, and cell viability was determined using the methylene blue staining assay. As demonstrated in Figure 3B, the presence of NAC prevented the decrease in cell viability induced by NRG. Two additional methods for detection of cell death (Hoecsht dye exclusion assay and flow cytometry) further supported these results. NRG induced enhanced cell death, as evident by the increase in sub-G1 population (Figure 3C) or by the high percentage of Hoecsht-positive cells (Figure 3D). This cell death was markedly inhibited by NAC. Furthermore, NAC treatment inhibited NRG-induced morphological change (S5). Hence, our findings clearly demonstrate that NAC inhibits the LC3-II accumulation, cell death and morphological changes induced by NRG in LNCaP cells.

Bottom Line:
It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death.Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

Methodology/principal findings: In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited.

Conclusions/significance: Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.